Dear students,

a classmate wrote a mail with some questions. I will reply here.

Please, write here, not by mail, to ask questions,

sincerely,

Giovanna

HI professor, goodnight.

I have a little problem in terms of IP and wb.

 Dear students,

I will try to answer here briefly, but I strongly suggest to watch the different videos about co-immunoprecipitations and western blot analysis. Moreover, please write your questions in the forum, not privately.

AS you said we use a primary antibody in IP to find the protein of interest then we wash the pellet with a buffer to eliminate protein A + beads.

No, we wash the pellet to eliminate proteins not bound to the primary antibody.

Remember that the beads are conjugated to protein A which is bound to primary antibody, which binds the target protein and the proteins bound to the target protein.

The beads are heavy: when you spin them, they go to the bottom of the tube. In the surnatant you will find only unbound proteins. You can eliminate them, you can add a washing solution, mix and spin again to completely eliminate unbound proteins.

After that we have SURNATANT containing protein of interest AND primary ANTIBODY.

No, in the SURNATANT you don’t have protein of interest and primary antibody. They are in the pellet, containing also agarose beads and protein A.

how we can eliminate the antibody before blotting.

You have to boil the immunoprecipitated proteins in the presence of a reducing agent, like beta-mercapto-ethanol and in the presence of the detergent SDS. The antibody detaches from the protein of interest and from the protein A. When you spin again, you will have in the bottom of the tube the sepharose beads, while you will have in solution the primary antibody and the protein of interest and the proteins bound to the protein of interest. The primary antibody runs in the gel together with your proteins.

Nevertheless, it is possible to bind covalently the primary antibody to the protein A; in that case, when you boil the sample, only the target protein and the proteins interacting with it will go in solution, the primary antibody remains attached to the protein A, bound to sepharose beads..

Another issue is that when we do WB we first add another primary antibody again than a secondary antibody /but between these 2 steps we should wash the pellet again? because you said if we wash, the enzymes which conjugate to ECL will be inactivated and so we should repeat all these steps with another secondary antibody to complete the wb 

I’m sorry, there is some confusion here.

After you boil your samples, you spin the agarose beads in the tube and you take the surnatant, containing the primary antibody (if not covalently bound to protein A), the protein of interest, and the proteins interacting with it. You have to load your samples in a gel, that you have to run and to blot on a membrane. Now you don’t have a pellet, you have a membrane with blotted proteins.

You add the primary antibody on the membrane, and you incubate it a few hours (overnight at 4 degrees or 2 hours at room temperature) then you wash the membrane to eliminate unbound antibody. Then, you have to add the secondary antibody, which is an antibody recognizing the primary antibody, conjugated with an enzyme, the Horse Radish Peroxidase (HRP). For example, if your primary antibody against your target protein is produced in mouse, the secondary antibody will be an antibody produced in another species (for example goat or donkey or sheep) recognizing the constant portion of all mouse antibodies. If your primary antibody is produced in rabbit, the secondary antibody will recognize the constant portion of all antibodies produced in rabbit. Then you have to add the substrate of the horse radish peroxidase.

I explained that sometime it is possible to wash well the membrane in the presence of sodium azide to inactivate the HRP, and you can add a new primary antibody to detect a new target protein. It is possible if the primary antibody recognizing the new protein is produced in a different animal, so the secondary antibody will recognize only the new antibody. Or it is possible to analyze different targets if their molecular weight is different, because you can identify them thanks to their molecular weight.

Please, contact me if you need further explanations.